Integrated Multi-omics Investigations Reveal the Key Role of Synergistic Microbial Networks in Removing Plasticizer Di-(2-Ethylhexyl) Phthalate from Estuarine Sediments

Di-(2-ethylhexyl) phthalate (DEHP) is the most widely used plasticizer worldwide, with an annual global production of more than 8 million tons. Because of its improper disposal, endocrine-disrupting DEHP often accumulates in estuarine sediments in industrialized countries at submillimolar levels, resulting in adverse effects on both ecosystems and human beings. The microbial degraders and biodegradation pathways of DEHP in O(2)-limited estuarine sediments remain elusive. Here, we employed an integrated meta-omics approach to identify the DEHP degradation pathway and major degraders in this ecosystem. Estuarine sediments were treated with DEHP or its derived metabolites, o-phthalic acid and benzoic acid. The rate of DEHP degradation in denitrifying mesocosms was two times slower than that of o-phthalic acid, suggesting that side chain hydrolysis of DEHP is the rate-limiting step of anaerobic DEHP degradation. On the basis of microbial community structures, functional gene expression, and metabolite profile analysis, we proposed that DEHP biodegradation in estuarine sediments is mainly achieved through synergistic networks between denitrifying proteobacteria. Acidovorax and Sedimenticola are the major degraders of DEHP side chains; the resulting o-phthalic acid is mainly degraded by Aestuariibacter through the UbiD-dependent benzoyl coenzyme A (benzoyl-CoA) pathway. We isolated and characterized Acidovorax sp. strain 210-6 and its extracellular hydrolase, which hydrolyzes both alkyl side chains of DEHP. Interestingly, genes encoding DEHP/mono-(2-ethylhexyl) phthalate (MEHP) hydrolase and phthaloyl-CoA decarboxylase—key enzymes for side chain hydrolysis and o-phthalic acid degradation, respectively—are flanked by transposases in these proteobacterial genomes, indicating that DEHP degradation capacity is likely transferred horizontally in microbial communities. IMPORTANCE Xenobiotic phthalate esters (PAEs) have been produced on a considerably large scale for only 70 years. The occurrence of endocrine-disrupting di-(2-ethylhexyl) phthalate (DEHP) in environments has raised public concern, and estuarine sediments are major DEHP reservoirs. Our multi-omics analyses indicated that complete DEHP degradation in O(2)-limited estuarine sediments depends on synergistic microbial networks between diverse denitrifying proteobacteria and uncultured candidates. Our data also suggested that the side chain hydrolysis of DEHP, rather than o-phthalic acid activation, is the rate-limiting step in DEHP biodegradation within O(2)-limited estuarine sediments. Therefore, deciphering the bacterial ecophysiology and related biochemical mechanisms can help facilitate the practice of bioremediation in O(2)-limited environments. Furthermore, the DEHP hydrolase genes of active DEHP degraders can be used as molecular markers to monitor environmental DEHP degradation. Finally, future studies on the directed evolution of identified DEHP/mono-(2-ethylhexyl) phthalate (MEHP) hydrolase would bring a more catalytically efficient DEHP/MEHP hydrolase into practice.